Mouse dermal fibroblasts express Crh and its receptors.

<p>(a) Total RNA isolated from whole brain from <i>Crh+/+</i> mice (lane 1), NMF isolated from <i>Crh+/+</i> mice (lane 2) and NMF isolated from <i>Crh−/−</i> mice (lane 3) was subjected to RT-PCR for evaluation of <i>Crh</i> expression. Lane 4 represents a sample without RT-enzyme and lane 5 represents a sample without template. (b) <i>Crf1</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from whole brain, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (c) <i>Crf2</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from heart, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (d) Effects of antalarmin and astressin-2B on specific [¹²⁵I] Tyr0-sauvagine binding to CRF₁ and CRF₂. Membrane homogenates from <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts were assayed for specific binding with [¹²⁵I] Tyr0-sauvagine, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a>. The bars represent the % decrease of specific binding. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. (e) Effect of CRH on cAMP accumulation in <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts. Stimulation of cAMP accumulation by CRH was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a> in intact cells. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. * represents statistical difference (P<0.05) between genotypes exposed to the same treatment and # represents statistical difference (P<0.05) between different treatments in the same genotype.</p

Enhanced migratory response of Crh−/− fibroblasts.

<p>Fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> dermis were allowed to attach on the dish. A clear space was produced in the confluent monolayer, cultured in serum free or serum treated with 10 µg/ml mytomycin and the wounded fibroblast layer was photographed immediately and 24 and 48 h after wounding. The distance of migration from the original borders was determined and is indicated (b) as a percentage of the original distance in a representative experiment. * represents statistical difference (P<0.05) between genotypes. (n = 3 wells/treatment/experiment, at least 4 independent experiments).</p

Proliferative response of Crh+/+ and Crh−/− fibroblasts.

<p>(a) Fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> dermis plated at an initial density of 8×10³ cells/well for four days. * represents statistical difference (P<0.05) between genotypes. (n = 4 wells/treatment/experiment, at least 5 independent experiments). (b) Fibroblasts isolated from both genotypes cultured at an initial density of 10⁵ cells/well in serum supplemented or serum free medium for 30 hr and apoptosis was measured by staining cells with Annexin V/Propidium Iodide and flow cytometry. The experiment was performed three times, with representative results of one experiment shown here.</p

IL-6 and TGF-β1 production from Crh+/+ and Crh−/− fibroblasts.

<p>Primary dermal fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> mice were cultured in 24-well plates (10⁵ cells/well). Supernatants were collected and analyzed by ELISA for IL-6 (a) or TGF-β1 (b). * represents statistical difference (P<0.05) between genotypes (n = 3 wells/condition/experiment, at least 3 independent experiments).</p

Effect of the CRH antagonists in human dermal fibroblasts.

<p>(a) HDFs isolated from human foreskin were cultured in 96-well plates (8,000 cells/well) in the presence of 100 nM antalarmin or ethanol (control). Proliferation was measured using either MTT or thymidine incorporation. *: represents statistical difference (P<0.05) between treatments (n = 4 wells/treatment/experiment, at least 3 independent experiments). (b) HDFs were allowed to attach on the 100 mm dish. A clear space was produced in the confluent monolayer, cultured in serum free or serum treated with 10 µg/ml mytomycin and the wounded fibroblast layer was photographed immediately and 24 h after wounding (n = 3 wells/treatment/experiment, at least 3 independent experiments). (c) HDFs were cultured in 24-well plates (10⁵ cells/well) in the presence of 100 nM antalarmin or ethanol (control). Supernatants were collected and analyzed by ELISA. * represents statistical difference (P<0.05) between treatments (n = 3 wells/condition/experiment, at least 3 independent experiments). (d) HDFs were cultured in 96-well plates (8,000 cells/well) in the presence of 1000 nM astressin 2B or ethanol (control). Proliferation was measured with MTT, (n = 4 wells/treatment/experiment, at least 3 independent experiments).</p

H₂S activates PKG and triggers vasodilatation.

<p>(A) Mouse aorta was incubated with NaHS (50 µM) for the indicated time and VASP phosphorylation on Ser239 was determined. Left: representative blot; right: quantitation of scanned autoradiograms, *p<0.05 vs vehicle, n = 3. (B) Incubation of aortic rings with the selective inhibitor of PKG, DT-2 (1, 3 µM) significantly inhibited NaHS-induced vasodilatation. TAT peptide (3 µM) was used as a control; *** p<0.001 vs. vehicle (dH₂O), n = 6 for each group. (C) Mice were injected with vehicle or DT-2 (100 nmoles, ip); after 15 min NaHS (1 µmol/kg) was administered subcutaneously. Systolic blood pressure (SBP) was monitored in conscious mice; *** p<0.001 vs vehicle, n = 4 for each group.</p

PKG contributes to the relaxing effect of exogenous and endogenous H₂S.

<p>(A) Mouse aortas from wild-type or PKG-I−/− animals were pre-treated with vehicle or glibenclamide (10 µM, 30 min) and then incubated with the indicated concentration of NaHS (n = 6 rings harvested from 3–4 animals); dashed lines are used for wild-type animals, while solid lines are used for knockouts. (B) L-cysteine-induced vasodilatation of aortic rings pre-contracted with phenylephrine from wild-type and PKG-I−/− mice. Note that cumulative concentration-response curves to L-cysteine were significantly different among the different strains of mice used CD1 vs 129/Sv (WT); °°° p<0.001 vs. CD1, *** p<0.001 vs WT, n = 8 rings harvested from 3–4 animals for each group (C) Representative blot and quantitation depicting aortic CSE expression in 129/Sv vs CD-1; n = 3 for each group, *p<0.05. (D) Representative blot showing expression of CSE in aortic homogenates in wild-type and PKG-I−/− mice. Experiments were performed twice with similar results.</p

Role of endothelium in H₂S induced-vasodilatation.

<p>(A) L-cysteine-induced vasodilatation was significantly impaired in aortic rings without endothelium (–end). (B) NaHS-induced vasodilatation is not affected by endothelium removal; *** p<0.001 vs –end, n = 6 for each group. (C) Representative photomicrographs of aortas stained with a CSE antibody and counter-stained for von-Willebrant factor, smooth muscle α-actin (SMA) and DAPI, showing localization of CSE.</p

GYY4137-induced relaxation is independent of PKG.

<p>Aortic smooth muscle cells were exposed to the indicated concentration of NaHS (A) or GYY4137 (B) and cGMP levels were determined after 5 min. *p<0.05 control, n = 4 for each group. (C) Incubation of isolated aortic rings with PKG selective inhibitor DT-2 (3 µM) did not affect GYY4137-induced vasodilatation; n = 6 for each group.</p